Compounds for the reducing lipotoxic damage

ABSTRACT

Provided herein are novel lipase inhibitors and methods for using the same to treat inflammation, multisystem organ failure, necrotic pancreatic acinar cell death, acute pancreatitis, sepsis (e.g., culture negative sepsis), burns, and acne. For example, provided herein are two novel lipase inhibitors useful in the methods described herein:or a pharmaceutically acceptable salt thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S.application Ser. No. 17/725,281, filed Apr. 20, 2022, now allowed, whichis a continuation of U.S. application Ser. No. 16/629,842, filed Jan. 9,2020, now U.S. Pat. No. 11,339,126, which is a National StageApplication under 35 U.S.C. § 371 that claims the benefit of ApplicationSerial No. PCT/US2018/041796, filed Jul. 12, 2018, which also claims thebenefit of U.S. Provisional Application Ser. No. 62/531,454, filed onJul. 12, 2017. The disclosures of the prior applications are consideredpart of (and are incorporated by reference in) the disclosure of thisapplication.

FIELD OF THE INVENTION

The present disclosure relates to the use of lipase inhibitors for thetreatment of severe pancreatitis and/or acne.

BACKGROUND OF THE INVENTION

The pancreas produces enzymes that aid in digestion and absorption offood; one such enzyme is lipase, which digests fat. Certain individuals(e.g., obese individuals) have an increased risk of developingmultisystem organ failure in acute inflammatory conditions such assevere burns, severe trauma, critical illness, and acute pancreatitis(AP). Pancreatitis is associated with the release of destructivedigestive enzymes from pancreatic acinar cells into the pancreas itself.When AP is initiated, it can quickly become severe AP (SAP). This is aconcern because SAP results in 40 to 50% mortality when complicated byacute renal failure, respiratory failure, hypocalcemia, and othermanifestations of multisystem organ failure or by large areas ofpancreatic necrosis. With no effective therapies, the current managementstandard is supportive care and managing complications when they occur.

SUMMARY OF THE INVENTION

Provided herein are novel lipase inhibitors and methods for using thesame to treat pancreatitis and/or organ failure and/or acne comprisingadministering, to a subject in need of such treatment, an effectiveamount of a lipase inhibitor as provided herein. The methods providedherein are based, at least in part, on the discoveries that lipotoxicitycontributes to inflammation, multisystem organ failure, necroticpancreatic acinar cell death, acute pancreatitis, sepsis (e.g., culturenegative sepsis), burns, acne, and infections, and that inhibition oflipase activity is able to reduce indices associated with theseconditions.

Accordingly, in some embodiments, provided herein are methods andcompositions for limiting lipotoxicity and thereby reducing thelikelihood of poor outcomes associated with acute pancreatitis and othersevere systemic conditions.

Provided herein is a compound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof. In some embodiments, thecompound is:

or a pharmaceutically acceptable salt thereof. In some embodiments, thecompound is:

or a pharmaceutically acceptable salt thereof.

Further provided herein is a pharmaceutical composition comprising oneor more of the compounds provided herein and a pharmaceuticallyacceptable excipient.

This disclosure also provides methods of treating acute pancreatitis ina subject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of a compound provided hereinor a pharmaceutically acceptable salt thereof. In some embodiments, theacute pancreatitis is severe. In some embodiments, the acutepancreatitis is downgraded from severe to mild following administration.In some embodiments, the subject is obese. In some embodiments, the riskof developing shock, renal failure, and/or pulmonary failure is reduced.

Also provided herein is a method of treating acne in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of a compound provided herein or apharmaceutically acceptable salt thereof.

Further provided herein is a method of treating sepsis in a subject inneed thereof, the method comprising administering to the subject atherapeutically effective amount of a compound provided herein or apharmaceutically acceptable salt thereof. In some embodiments, thesepsis is culture negative sepsis.

This disclosure also provides methods of treating burns in a subject inneed thereof, the method comprising administering to the subject atherapeutically effective amount of a compound provided herein or apharmaceutically acceptable salt thereof.

Also provided herein is a method for treating infections in a subject inneed thereof, the method comprising administering to the subject atherapeutically effective amount of a compound provided herein or apharmaceutically acceptable salt thereof. In some embodiments, theinfection is caused by one or more organisms selected from the groupconsisting of P. auregenosa, S. aureus, B. subtilis, and B. cepecia.

In some of the above embodiments, the compound is:

or a pharmaceutically acceptable salt thereof. In some embodiments ofthe above embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the structure of orlistat dividing the compoundinto three main regions.

FIG. 2 is a scheme detailing a synthetic route to prepare amino-estermodifications to the orlistat core.

FIG. 3 shows the structures of the various non-formylated amino estermodified analog compounds prepared.

FIGS. 4A-4B provide line graphs showing the results of a lipaseinhibition assay testing the non-formylated amino ester modified analogcompounds. FIG. 4A provides the results of freshly prepared solutions ofthe compounds while FIG. 4B provides the results of solutions storedovernight.

FIG. 5 is a scheme detailing a synthetic route to prepare formulatedamino ester modified analogs.

FIG. 6 shows the structures of the various formyl amino ester modifiedanalog compounds prepared.

FIGS. 7A-7B provide line graphs showing the results of a lipaseinhibition assay testing the formyl amino ester modified analogcompounds. FIG. 7A provides the results of freshly prepared solutions ofthe compounds while FIG. 7B provides the results of solutions storedovernight.

FIGS. 8A-8B provide bar graphs detailing the efficacy of selected analogcompounds in reducing lipotoxic damage to acinar cells inacinar-adipocyte co-culture (FIG. 8A) while also assessing the toxicityof the compounds to these cells (FIG. 8B).

FIG. 9 is a scheme detailing the metastasis reaction used to prepare theβ-chain analogs.

FIG. 10 is a scheme detailing a synthetic route to the β-chain analogs.

FIG. 11 is a scheme detailing a synthetic route to the β-chain analogs.

FIG. 12 is a scheme detailing a synthetic route to the β-chain analogs.

FIG. 13 is a scheme detailing a synthetic route to the β-chain analogs.

FIG. 14 shows the structures of the various β-chain analogs prepared.

FIG. 15 provides a line graph showing the results of a lipase inhibitionassay testing the β-chain analogs in freshly prepared solutions.

FIG. 16 is a scheme detailing a synthetic route to the α-chain analogs.

FIGS. 17A-17B provide line graphs showing the results of a lipaseinhibition assay testing the α-chain analogs. FIG. 17A provides theresults of freshly prepared solutions of the compounds while FIG. 17Bprovides the results of solutions stored overnight.

FIGS. 18A-18B provide line graphs showing the results of an assayexploring % free fatty acid (FFA) generation. FIG. 18A provides theresults of freshly prepared solutions of the compounds while FIG. 18Bprovides the results of solutions stored overnight.

FIG. 19 shows tables of values obtained from toxicity studies in mousepancreatic cells.

FIGS. 20A-20B provide line graphs showing the results of an assayexploring % FFA generation. FIG. 20A provides the results with hPLRP2lipase while FIG. 20B provides the results with hCEL lipase.

FIG. 21 provides a tabular summary of the in vitro studies describedherein.

FIG. 22 provides a summary of the use of orlistat in reducing severepancreatitis.

FIGS. 23A-23G provide the results of in vivo testing of compound 767.FIG. 23A provides the study timeline; FIG. 23B provides a bar graph ofthe serum lipase concentration;

FIG. 23C provides a graph showing serum blood urea nitrogen levels atvarious time points;

FIG. 23D provides a graph showing serum calcium levels at various timepoints; and FIG. 23E provides a graph showing protection againstsystemic inflammatory response syndrome (SIRS); FIG. 23F provides agraph showing protection against shock and FIG. 23G provides a graphshowing improved survival

FIG. 24 provides photographs of treated mice.

FIG. 25 provides a tabular summary of the in vivo studies with compound767 and orlistat.

FIG. 26 provides a line graph showing % FFA generation in a medium of P.acnes.

FIG. 27 provides pictures of spirit blue agar inoculated with P. acnesand exposed to both orlistat and compound 767.

FIG. 28 shows efficacy of 10 mM agents in reducing LDH leakage fromacinar cells incubated with 600 mM GTL.

DETAILED DESCRIPTION OF THE INVENTION

Presently, severe pancreatitis is often treated through administrationof the FDA approved lipase inhibitor, orlistat. While lipase inhibitionreduces the severity of pancreatitis, the use of orlistat is complicatedby the need of repeated dosing (e.g., 50 mg/kg BID for two days) duringpancreatitis, and the hypertriglyceridemia associated with the use ofthis compound. Provided herein are compounds that provide therapeuticbenefits in pancreatitis, without the need of repeated dosing or thecomplications associated with orlistat.

Compounds provided herein include those described in Table 1.

TABLE 1 Compound Number Structure 728

731

729

727

725

716

718

717

726

724

732

733

734

738

736

737

741

739

740

743

742

762

763

760

767

768

or a pharmaceutically acceptable salt thereof. In some embodiments, thecompound is

or a pharmaceutically acceptable salt thereof.

In some embodiments, a compound provided herein is stable in aqueoussolution. For example, a compound provided herein can be more stablethan orlistat in aqueous solution. In some embodiments, the compound is

or a pharmaceutically acceptable salt thereof. In some embodiments, thecompound is

or a pharmaceutically acceptable salt thereof.

Synthesis

The compounds described herein can be prepared, for example, accordingto the procedures described in the Examples and associated figures.

It will be appreciated by one skilled in the art that the processesdescribed are not the exclusive means by which compounds provided hereinmay be synthesized and that a broad repertoire of synthetic organicreactions is available to be potentially employed in synthesizingcompounds provided herein. The person skilled in the art knows how toselect and implement appropriate synthetic routes. Suitable syntheticmethods of starting materials, intermediates and products may beidentified by reference to the literature, including reference sourcessuch as: Advances in Heterocyclic Chemistry, Vols. 1-107 (Elsevier,1963-2012); Journal of Heterocyclic Chemistry Vols. 1-49 (Journal ofHeterocyclic Chemistry, 1964-2012); Carreira, et al. (Ed.) Science ofSynthesis, Vols. 1-48 (2001-2010) and Knowledge Updates KU2010/1-4;2011/1-4; 2012/1-2 (Thieme, 2001-2012); Katritzky, et al. (Ed.)Comprehensive Organic Functional Group Transformations, (Pergamon Press,1996); Katritzky et al. (Ed.); Comprehensive Organic Functional GroupTransformations II (Elsevier, 2^(nd) Edition, 2004); Katritzky et al.(Ed.), Comprehensive Heterocyclic Chemistry (Pergamon Press, 1984);Katritzky et al., Comprehensive Heterocyclic Chemistry II, (PergamonPress, 1996); Smith et al., March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, 6^(th) Ed. (Wiley, 2007); Trost etal. (Ed.), Comprehensive Organic Synthesis (Pergamon Press, 1991).

The reactions for preparing compounds described herein can be carriedout in suitable solvents which can be readily selected by one of skillin the art of organic synthesis. Suitable solvents can be substantiallynon-reactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,(e.g., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature). A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected by the skilled artisan.

Preparation of compounds described herein can involve the protection anddeprotection of various chemical groups. The need for protection anddeprotection, and the selection of appropriate protecting groups, can bereadily determined by one skilled in the art. The chemistry ofprotecting groups can be found, for example, in T. W. Greene and P. G.M. Wuts, Protective Groups in Organic Synthesis, 3^(rd) Ed., Wiley &Sons, Inc., New York (1999).

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or¹³C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), massspectrometry, or by chromatographic methods such as high performanceliquid chromatography (HPLC), liquid chromatography-mass spectroscopy(LCMS), or thin layer chromatography (TLC). Compounds can be purified bythose skilled in the art by a variety of methods, including highperformance liquid chromatography (HPLC) and normal phase silicachromatography.

The term “compound” as used herein is meant to include allstereoisomers, geometric isomers, tautomers, and isotopes of thestructures depicted. Compounds herein identified by name or structure asone particular tautomeric form are intended to include other tautomericforms unless otherwise specified.

Compounds provided herein also include tautomeric forms. Tautomericforms result from the swapping of a single bond with an adjacent doublebond together with the concomitant migration of a proton. Tautomericforms include prototropic tautomers which are isomeric protonationstates having the same empirical formula and total charge. Exampleprototropic tautomers include ketone—enol pairs, amide—imidic acidpairs, lactam—lactim pairs, enamine—imine pairs, and annular forms wherea proton can occupy two or more positions of a heterocyclic system, forexample, 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be inequilibrium or sterically locked into one form by appropriatesubstitution.

In some embodiments, the compounds described herein can contain one ormore asymmetric centers and thus occur as racemates and racemicmixtures, enantiomerically enriched mixtures, single enantiomers,individual diastereomers and diastereomeric mixtures (e.g., including(R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (+)(dextrorotatory) forms, (−) (levorotatory) forms, the racemic mixturesthereof, and other mixtures thereof). Additional asymmetric carbon atomscan be present in a substituent, such as an alkyl group. All suchisomeric forms, as well as mixtures thereof, of these compounds areexpressly included in the present description. The compounds describedherein can also or further contain linkages wherein bond rotation isrestricted about that particular linkage, e.g. restriction resultingfrom the presence of a ring or double bond (e.g., carbon-carbon bonds,carbon-nitrogen bonds such as amide bonds). Accordingly, all cis transand E/Z isomers and rotational isomers are expressly included in thepresent description. Unless otherwise mentioned or indicated, thechemical designation of a compound encompasses the mixture of allpossible stereochemically isomeric forms of that compound.

Optical isomers can be obtained in pure form by standard proceduresknown to those skilled in the art, and include, but are not limited to,diastereomeric salt formation, kinetic resolution, and asymmetricsynthesis. See, for example, Jacques, et al., Enantiomers, Racemates andResolutions (Wiley Interscience, New York, 1981); Wilen, S. H., et al.,Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistry of CarbonCompounds (McGraw-Hill, N Y, 1962); Wilen, S. H. Tables of ResolvingAgents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of NotreDame Press, Notre Dame, Ind. 1972), each of which is incorporated hereinby reference in their entireties. It is also understood that thecompounds described herein include all possible regioisomers, andmixtures thereof, which can be obtained in pure form by standardseparation procedures known to those skilled in the art, and include,but are not limited to, column chromatography, thin-layerchromatography, and high-performance liquid chromatography.

Unless specifically defined, compounds provided herein can also includeall isotopes of atoms occurring in the intermediates or final compounds.Isotopes include those atoms having the same atomic number but differentmass numbers. Unless otherwise stated, when an atom is designated as anisotope or radioisotope (e.g., deuterium, [¹¹C], [¹⁸F]), the atom isunderstood to comprise the isotope or radioisotope in an amount at leastgreater than the natural abundance of the isotope or radioisotope. Forexample, when an atom is designated as “D” or “deuterium”, the positionis understood to have deuterium at an abundance that is at least 3000times greater than the natural abundance of deuterium, which is 0.015%(i.e., at least 45% incorporation of deuterium).

All compounds, and pharmaceutically acceptable salts thereof, can befound together with other substances such as water and solvents (e.g.hydrates and solvates) or can be isolated.

In some embodiments, preparation of compounds can involve the additionof acids or bases to affect, for example, catalysis of a desiredreaction or formation of salt forms such as acid addition salts.

Example acids can be inorganic or organic acids and include, but are notlimited to, strong and weak acids. Some example acids includehydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,p-toluenesulfonic acid, 4-nitrobenzoic acid, methanesulfonic acid,benzenesulfonic acid, trifluoroacetic acid, and nitric acid. Some weakacids include, but are not limited to acetic acid, propionic acid,butanoic acid, benzoic acid, tartaric acid, pentanoic acid, hexanoicacid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid.

Example bases include lithium hydroxide, sodium hydroxide, potassiumhydroxide, lithium carbonate, sodium carbonate, potassium carbonate, andsodium bicarbonate. Some example strong bases include, but are notlimited to, hydroxide, alkoxides, metal amides, metal hydrides, metaldialkylamides and arylamines, wherein; alkoxides include lithium, sodiumand potassium salts of methyl, ethyl and t-butyl oxides; metal amidesinclude sodium amide, potassium amide and lithium amide; metal hydridesinclude sodium hydride, potassium hydride and lithium hydride; and metaldialkylamides include lithium, sodium, and potassium salts of methyl,ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, trimethylsilyl andcyclohexyl substituted amides.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The present application also includes pharmaceutically acceptable saltsof the compounds described herein. As used herein, “pharmaceuticallyacceptable salts” refers to derivatives of the disclosed compoundswherein the parent compound is modified by converting an existing acidor base moiety to its salt form. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. The pharmaceuticallyacceptable salts of the present application include the conventionalnon-toxic salts of the parent compound formed, for example, fromnon-toxic inorganic or organic acids. The pharmaceutically acceptablesalts of the present application can be synthesized from the parentcompound which contains a basic or acidic moiety by conventionalchemical methods. Generally, such salts can be prepared by reacting thefree acid or base forms of these compounds with a stoichiometric amountof the appropriate base or acid in water or in an organic solvent, or ina mixture of the two; generally, non-aqueous media like ether, ethylacetate, alcohols (e.g., methanol, ethanol, iso-propanol, or butanol) oracetonitrile (MeCN) are preferred. Lists of suitable salts are found inRemington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company,Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2(1977). Conventional methods for preparing salt forms are described, forexample, in Handbook of Pharmaceutical Salts: Properties, Selection, andUse, Wiley-VCH, 2002.

Pharmaceutical Compositions

When employed as pharmaceuticals, the compounds provided herein andpharmaceutically acceptable salts thereof can be administered in theform of pharmaceutical compositions. These compositions can be preparedas described herein or elsewhere, and can be administered by a varietyof routes, depending upon whether local or systemic treatment is desiredand upon the area to be treated. Administration may be topical(including transdermal, epidermal, ophthalmic and to mucous membranesincluding intranasal, vaginal and rectal delivery), pulmonary (e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal or intranasal), oral, or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal intramuscular or injection or infusion; or intracranial,(e.g., intrathecal or intraventricular, administration). Parenteraladministration can be in the form of a single bolus dose, or may be, forexample, by a continuous perfusion pump. In some embodiments, thecompounds provided herein, or a pharmaceutically acceptable saltthereof, are suitable for parenteral administration. In someembodiments, the compounds provided herein are suitable for intravenousadministration. In some embodiments, the compounds provided herein aresuitable for oral administration. In some embodiments, the compoundsprovided herein are suitable for topical administration.

Pharmaceutical compositions and formulations for topical administrationmay include, but are not limited to, transdermal patches, ointments,lotions, creams, gels, drops, suppositories, sprays, liquids andpowders. Conventional pharmaceutical carriers, aqueous, powder or oilybases, thickeners and the like may be necessary or desirable. In someembodiments, the pharmaceutical compositions provided herein aresuitable for parenteral administration. In some embodiments, thecompounds provided herein can be formulated for topical delivery (e.g.,for the treatment of acne). An exemplary formulation includes:

about 10 to about 50 mM of a compound provided herein (e.g., a compound767)

about 50% (v/v) 99.5-100% ethanol or isopropanol

about 0.25 to about 2% (w/v) carbomer (e.g, carbomer 934 or carbomer940)

about 0.5 to about 1% (w/v) trolamine

water (to 100%).

In some embodiments, the pharmaceutical compositions provided herein aresuitable for intravenous administration. In some embodiments, thepharmaceutical compositions provided herein are suitable for oraladministration. In some embodiments, the pharmaceutical compositionsprovided herein are suitable for topical administration.

Also provided are pharmaceutical compositions which contain, as theactive ingredient, a compound provided herein, or a pharmaceuticallyacceptable salt thereof, in combination with one or morepharmaceutically acceptable carriers (e.g. excipients). In making thepharmaceutical compositions provided herein, the active ingredient istypically mixed with an excipient, diluted by an excipient or enclosedwithin such a carrier in the form of, for example, a capsule, sachet,paper, or other container. When the excipient serves as a diluent, itcan be a solid, semi-solid, or liquid material, which acts as a vehicle,carrier or medium for the active ingredient. Thus, the compositions canbe, for example, in the form of tablets, pills, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,aerosols (as a solid or in a liquid medium), ointments, soft and hardgelatin capsules, suppositories, sterile injectable solutions, andsterile packaged powders.

Some examples of suitable excipients include, without limitation,lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water,syrup, and methyl cellulose. The formulations can additionally include,without limitation, lubricating agents such as talc, magnesium stearate,and mineral oil; wetting agents; emulsifying and suspending agents;preserving agents such as methyl- and propylhydroxy-benzoates;sweetening agents; flavoring agents, or combinations thereof.

The active compound can be effective over a wide dosage range and isgenerally administered in an effective amount. It will be understood,however, that the amount of the compound actually administered willusually be determined by a physician, according to the relevantcircumstances, including the condition to be treated, the chosen routeof administration, the actual compound administered, the age, weight,and response of the individual subject, the severity of the subject'ssymptoms, and the like.

The compositions provided herein can be administered one from one ormore times per day to one or more times per week; including once everyother day. The skilled artisan will appreciate that certain factors caninfluence the dosage and timing required to effectively treat a subject,including, but not limited to, the severity of the disease or disorder,previous treatments, the general health and/or age of the subject, andother diseases present.

Moreover, treatment of a subject with a therapeutically effective amountof a compound described herein can include a single treatment or aseries of treatments. In some embodiments, the compositions providedherein are administered as a single dose.

Dosage, toxicity and therapeutic efficacy of the compounds providedherein can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., for determining the LD₅₀ (thedose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD₅₀/ED₅₀. Compounds exhibiting hightherapeutic indices are preferred. While compounds that exhibit toxicside effects can be used, care should be taken to design a deliverysystem that targets such compounds to the site of affected tissue inorder to minimize potential damage to uninfected cells and, thereby,reduce side effects. In some embodiments, the compounds provided hereinexhibit lower toxicity as compared to similar dosages of orlistat.Evaluation of toxicity may be determined, for example, using methodssuch as those described herein.

Methods of Treatment

The present disclosure further provides methods for the treatment ofdisorders associated with elevated lipid concentrations (e.g.,lipotoxicity). A number of such disorders are known in the art and canbe readily identified by one of skill in the art. In some embodiments,the methods include a method for inhibiting serum lipases in a subjectin need thereof, the method comprising administering to the subject atherapeutically effective amount of a compound provided herein, or apharmaceutically acceptable salt thereof. In some embodiments, thecompounds provided herein decrease serum lipase concentrations. In someembodiments, the lipase is a pancreatic lipase such as human pancreatictri-acyl glycerol lipase (hPNLIP), human pancreatic lipase relatedprotein 2 (hPLRP2) and human colipase (hCEL). In some embodiments, thecompounds provided herein can reduce or inhibit generation of free fattyacids. For example, the compounds provided herein can decrease serumconcentrations of free fatty acids.

As used herein, the term “subject,” refers to any animal, includingmammals. For example, the term “subject” includes, but is not limitedto, mice, rats, other rodents, rabbits, dogs, cats, swine, cattle,sheep, horses, primates, and humans. In some embodiments, the subject isa human. In some embodiments, the subject is obese. The term “obese” asused herein refers to a subject having a body mass index of greater than23, 25 and particularly greater than or equal to 30. Alternatively itcould an increase in abdominal fat or girth more than normal for therace, ethnicity, sex or nationality of the individual.

In some embodiments, the methods described herein can include in vitromethods, e.g., contacting a sample (e.g., a cell or tissue) with acompound provided herein, or a pharmaceutically acceptable salt thereof.

In some embodiments, the disorder is acute pancreatitis. For example,the acute pancreatitis can be severe. In some embodiments, the methodsprovided herein can be useful in downgrading a severe case of acutepancreatitis to a mild case of pancreatitis. The disclosure providesmethods of treating one or more symptoms of pancreatitis in a subject inneed thereof by administering a therapeutically effective amount of acompound provided herein, or a pharmaceutically acceptable salt thereof,to treat one or more symptoms of acute pancreatitis. Exemplary symptomsof acute pancreatitis include abdominal pain, back pain, swollenabdomen, nausea, vomiting, fever, rapid pulse, shortness of breath, lowbody temperature and the like.

Non-limiting embodiments of the invention provide for a method ofreducing the risk of organ failure in a subject suffering from acutepancreatitis comprising administering, to the subject, an effectiveamount of a pancreatic lipase inhibitor. Organs for which the risk oforgan failure may be reduced include the kidney (where failure isreferred to as renal failure), the lung (where failure is referred to aspulmonary failure), Shock with low blood pressure or an increase inheart rate or the appearance of pulmonary edema (noted as crackles onauscultation, or CXR, or CT scan) as well as multisystem organ failure(e.g. multiple organ dysfunction syndrome involving at least these twoorgans). The status of these organs may be determined using clinicalmethods well known in the art. For example, and not by way oflimitation, kidney function (and the development of renal failure) maybe assessed via increased blood urea nitrogen levels, increasedcreatinine, decreased urine output, and/or histologic findings; lungfunction and the development of pulmonary failure may be assessed usingpulmonary function tests, blood gases (oxygen and carbon dioxidelevels), oxygen supplementation requirements (e.g. nasal cannula or facemask or ventilator, with different percentages and flow rates of oxygen)and/or histologic findings; (for indices of organ failure, see J.Wallach, 1978, Interpretation of Diagnostic Tests, Third Edition,Little, Brown and Co., Boston, and/or J. Wallach, 2006, Interpretationof Diagnostic Tests, Eighth Edition, Lippincott Williams & Wilkins, bothincorporated by reference in their entireties). Likewise, an index ofsystemic inflammation is an increase in levels of one or moreinflammatory mediators, including but not limited to CRP, or parts ofsystemic inflammatory response syndrome (SIRS) criteria, tumor necrosisfactor alpha, monocyte chemotactic protein 1 and/or interleukin 6. Theseembodiments are supported, at least in part, by working examples below,which show the effectiveness of pancreatic lipase inhibitors indecreasing the risk of multisystem organ failure.

In some embodiments, the compounds provided herein are useful forreducing the risk of secondary effects of acute pancreatitis in asubject in need thereof. For example, the risk of developing shock canbe reduced. In some embodiments, the risk of developing renal failure isreduced. In some embodiments, the risk of developing pulmonary failureis reduced.

In some embodiments, the disorder is acne. In other embodiments, thedisorder is trauma, hemorrhage, critical illness, or sepsis. Forexample, the sepsis is culture negative sepsis. In some embodiments, thedisorder is a burn. In other embodiments, the disorder is an infection.For example, the infection may be caused by one or more organismsselected from the group consisting of P. auregenosa, S. aureus, B.subtilis, and B. cepecia. See, for example, Ryan C M, Sheridan R L,Schoenfeld D A, Warshaw A L, Tompkins R G: Postburn pancreatitis. AnnSurg 1995, 222(2):163-170; Subramanian A, Albert V, Mishra B, Sanoria S,Pandey R M: Association Between the Pancreatic Enzyme Level and OrganFailure in Trauma Patients. Trauma Mon 2016, 21(2):e20773; Malinoski DJ, Hadjizacharia P, Salim A, Kim H, Dolich M O, Cinat M, Barrios C,Lekawa M E, Hoyt D B: Elevated serum pancreatic enzyme levels afterhemorrhagic shock predict organ failure and death. J Trauma 2009,67(3):445-449; Lee C C, Chung W Y, Shih Y H: Elevated amylase and lipaselevels in the neurosurgery intensive care unit. J Chin Med Assoc 2010,73(1):8-14; and Manjuck J, Zein J, Carpati C, Astiz M: Clinicalsignificance of increased lipase levels on admission to the ICU. Chest2005, 127(1):246-250.

EXAMPLES Example 1. Preparation and Testing of Non-Formylated Amino AcidAnalogs

A number of non-formylated amino acid analogs were prepared in which theL-amino-ester leucine was replaced by alternates. The synthetic stepsinvolved in this process are outlined in FIG. 2 . As shown in FIG. 3 ,the compounds generated included compounds where the L-leucine (#723,which is also orlistat) amino-ester was replaced by alternates such asN-methyl leucine (#724) or more polar or shorter alternates, includingL-alanine (#716), N-acetyl-L-cystine (#717), L-aspartate (#718),L-histidine (#725), L-methionine sulfone (#726), L-arginine (727),L-diaminopimelic acid (#728), L-serine (#729), L-ornithine (#731), orN-acetyl leucine (#732).

To test the efficacy of these compounds, the agents were dissolved as a20 mM stock in ethanol, and further diluted to in PBS as a 600micromolar working stock (3.3% ethanol). This was either usedimmediately or left at room temperature for 16-20 hours overnight beforeretesting efficacy. The agents were incubated with a pancreatic lysatein serial 1/3 dilutions spanning a range of 200 to 0.3 micromolars for30 minutes. 5 microliters of these mixtures were then used to measurelipase activity using a commercial lipase assay (Pointe scientific)containing a monoacyl glycerol lipase in the reagent. The lipase assaywas done using reagents in proportion to the recommended protocol.Values were depicted as % maximal lipase activity of the well withoutany inhibitor. As can be seen there was no increase in efficacy of thenon-formylated orlistat analogs over that of orlistat (FIGS. 4A-B).

Example 2. Preparation and Testing of Formylated Amino Acid Analogs

A number of formylated amino acid analogs were prepared in which theL-amino-ester leucine was replaced by alternates. The synthetic stepsinvolved in this process are outlined in FIG. 2 and FIG. 5 .

These agents (#733, 734, 738, 739, 740, 742, 743, as shown in FIG. 6 )were initially tested as described in Example 1. It was noted thatcompounds 741, 736, 743 had an approximately 10-fold improvement instability and efficacy over orlistat after overnight storage (FIGS.7A-B). The compounds were further tested for efficacy in reducinglipotoxic damage to acinar cells in acinar-adipocyte co-culture asdescribed in Navina S, Acharya C, DeLany J P, Orlichenko L S, Baty C J,Shiva S S, Durgampudi C, Karlsson J M, Lee K, Bae K T et al:Lipotoxicity causes multisystem organ failure and exacerbates acutepancreatitis in obesity. Sci Transl Med 2011, 3(107):107ra110 (FIG. 8A),and while all agents were effective at 66 micromolars (the nearestconcentration to IC₅₀ of orlistat after overnight incubation), compound743 was noted to be toxic to acinar cells (FIG. 8B) and compound 741 wasthe least toxic at this concentration, while retaining its efficacy.Compound 736 also retained its efficacy at low toxicities.

Example 3. Preparation and Testing of β-Chain Analogs

To further explore options for improving efficacy of the analogs, thebeta chain of 741 was altered to make it more hydrophilic. For this,compounds 760, 762, 763 were prepared as described in FIGS. 9-14 . Themodifications at this position were associated with no improvement inthe lipase inhibition efficacy, and, in fact, a decrease in efficacy wasobserved. See FIG. 15 .

Example 4. Preparation and Testing of α-Chain Analogs

A series of analogs were prepared based on modifications to the alphachain as shown in FIG. 16 , generating compounds 767 and 768. Whilethese agents had equivalent efficacy in the lipase activity assay toorlistat when fresh, agent 767 significantly retained its efficacy as asolution when left overnight. See FIGS. 17A-B.

Example 5. Inhibition of Free Fatty Acid (FFA) Generation, Toxicity, andEfficacy in Inhibiting Recombinant Human Pancreatic Tri-Acyl GlycerolLipase [hPNLIP], Pancreatic Lipase Related Protein 2 (hPLRP2) andCarboxyester Lipase (hCEL)

The efficacy of a selection of the prepared analogs was explored todetermine their ability to inhibit free fatty acid (FFA) generation fromrecombinant human lipases while retaining stability. To test thesefeatures, the analogs were dissolved in ethanol (200 mM stock) andfurther diluted to a 600 micromolar (0.3% ethanol) working stock in PBS.The agents were incubated with the recombinant enzymes in serial 1/3dilutions spanning a range of 200 to 0.3 micromolar for 30 minutes, andthen added to the substrate 1 mM glyceryl trilinoleate (GTL) in PBS, pH7.4 along with cofactor colipase (CLPS; 0.5 mcg/ml). As seen in FIGS.18A-B, agents 767, 733, 734, 740 and 743 had IC₅₀s against recombinanthuman pancreatic tri-acyl glycerol lipase [PNLIP; hPTL, in the presenceof colipase (CLPS)] that were 50× lower than orlistat or 741, whileretaining an IC₅₀<2 micromolar when stored overnight. In particular,compounds 767 and 740 displayed the best stability.

Toxicity studies of the relevant compounds were also performed using bydiluting the original ethanol stock 200 mM into the incubation mediumfor acinar cells (HEPES buffer, pH 7.4, as described by Navina et al)600 micromolar stock solutions of the compounds in 0.3% ethanol in PBS.These were added to acinar cells at 1/3 dilutions starting at 200micromolar. Mouse pancreatic acinar ATP levels and LDH leakage weremeasured over 4 hours and compared to control. Any number positive overcontrol indicates % injury greater than control acini. Agent 743 (SeeFIG. 8B), and 733 were, however, toxic to acinar cells, causing a dropin ATP and inducing LDH leakage (see FIG. 19 ) at concentrations in the20-200 micromolar range-concentrations relevant to in vivo use.

These compounds were further tested for efficacy in reducing recombinanthuman pancreatic lipase related protein 2 (hPLRP2) and carboxyesterlipase (hCEL) as shown in FIGS. 20A-B. These lipases form 15-30% ofpancreatic lipases. Agents 740, 734 and 767 again exhibited IC₅₀s of 2micromolars or less. The relative potency and toxicities are summarizedin the table in FIG. 21 . Noting this, along with the strengths of 767,i.e., a) the higher efficacy against mouse pancreatic lysates (FIGS.7A-B and 17), b) higher potency against the most prevalent lipase (hPTL)(FIGS. 18A-B), c) better stability in aqueous media (FIGS. 17A-B and18), and d) lower toxicity (FIG. 19 ), compound 767 was elected toproceed on to in vivo studies. Furthermore, Agents 740 and 767 at 10micromolar were less toxic and more efficacious in reducing GTL inducedacinar injury than orlistat (FIG. 28 )

Example 6. In Vivo Studies of Compound 767

The goal of this in vivo study was to learn if the severity of lethalpancreatitis could be reduced in mice when compound 767 is administeredas a single therapeutic dose, and whether the compound could avoid thelimitations of orlistat, e.g., repeated high dosing andhypertriglyceridemia. See FIG. 22 .

Compound 767 or orlistat was dissolved as 1 mg/10 microliter ethanol,which was diluted to 400 microliters with saline (final concentration2.5% ethanol). Genetically obese male ob/ob mice (50-60 gm) were usedand baseline tail vein blood draw was collected for serum lipase,triglycerides, and BUN (blood urea nitrogen). The time line is shown asin FIG. 23A. Severe pancreatitis was induced by implanting an alzet pumpsubcutaneously containing 2.5 mg/ml caerulein, which was administered ata dose of 50 mc/kg/hr. Six hours later a tail vein sample (50microliters) was tested for serum lipase to verify an increase >3 foldbasal. As seen in FIG. 23B, this was achieved (P=0.003).

The agents were then administered as an intraperitoneal dose (20 mg/kgsingle dose [n=8 for 767 or orlistat n=7]. Animals were given saline IP1 mL BID, vitals were monitored daily focusing on severity (Temperaturefor fever or hypothermia and Pulse distention for shock). These arerespectively part of SIRS and revised Atlanta criteria for severity(Classification of acute pancreatitis—2012: revision of the Atlantaclassification and definitions by international consensus. Gut 2013,62(1):102-111). tail vein samples were collected for severity parameters(Serum BUN, serum calcium and total white blood cell count) which are apart of Ranson's and Glasgow criteria) and triglycerides. Animals weresacrificed if they were moribund.

As can be seen in FIGS. 23A-G, in vivo all untreated mice developedrenal failure (BUN 85±18 mg/dl) FIG. 23C, hypocalcemia (serum calcium4.9±0.2 mg/dl) FIG. 23D, SIRS FIG. 23E, shock FIG. 23F and 100%mortality. FIG. 23G. 767 was more efficacious than orlistat innormalizing BUN (peak 18.5±0.9 vs. orlistat 50±4.4 mg/dl, p<0.05) FIG.23C, serum calcium (8.8±1.0 mg/dl vs. 4.2±0.3 mg/dl, p<0.05) FIG. 23D,UFAs (2.4±0.4 mM vs. 5.4±0.9 mM), preventing shock (PD: 290±20 μm vs.171±21 μm, p<0.05) FIG. 23F, leucopenia (in 0/8 mice vs. 4/7mice,p<0.03, see FIG. 25 ), hypothermia (94.1±0.8 vs. 86±0.9° F., p<0.01;FIG. 23E) and improving 5 day survival (8/8 vs. 2/7, p<0.01) FIG. 23G.There was 8/8 (meantime 44 hours) mortality in the vehicle group, 5/7mortality in the orlistat group. The gross appearance with the twoagents is compared in FIG. 24 . As can be seen there is much moreextensive fat necrosis in the orlistat treated animal. This protectionfrom agent 767 was achieved without the hypertriglyceridemia associatedwith the dosing needed for orlistat to be protective (FIG. 25 ). Theresults are summarized in the table in FIG. 25 .

Example 7. FFA Generation in a Medium of P. acnes

The lipolysis by P. acnes was inhibited by 767 (FIG. 26 and FIG. 27 ).

P. acnes BROTH Culture:

For medium, 2.0 g of Casein hydrolysate enzymatic digest (Cat #12855,Affymetrix Inc, Ohio, USA), 1.0 g of Yeast extract (Cat #61180-5000,Acros organics, New Jersey, USA) and 0.03 g of Victoria Blue B (Cat#sc-216055, Santa Crutz Biotechnology Inc, California, USA) weredissolved completely in 200 ml of double distilled water with mildheating. The mixed solution was sterilized by autoclaving at 15 lbspressure (121° C.) for 15 min. The autoclaved solution allowed to cooldown to 50° C., then 6 ml of emulsified lipase substrate slowly addedwith continuous stirring to get an even distribution and stored at 4° C.for P. acnes culture.

Lipase substrate preparation: Lipase substrate was prepared by mixing200 μl of Tween 80 (Cat #P4780, Sigma, St louis, USA) in 100 ml of warmdouble distilled water and 25 ml of Cotton seeds oil (Cat #C0145,Spectrum chemical, California, USA). The mixture was agitated vigorouslyfollowed by sonication (10 sec pulse×5 times) to get an emulsion.

P. acnes culture-expansion medium, storage conditions, size of inoculumused in medium.

A small part of lyophilized Propionibacterium acnes (Strain VPI0389,ATCC) was added to the medium and allowed to grow at 37° C. for 48 h ina BD GasPak EZ standard incubation container (Cat #260671, BecktonDickinson, Md., USA) with two anaerobe Sachets (Cat #260683, BecktonDickinson, Md., USA) which maintained the anaerobic condition within thecontainer. After 48 h of culture, bacterial count was measured byturbidity (OD600 nm) method. Amount of secreted lipase and produced FFAwere measure in the culture supernatant.

For P. acnes storage, cultured bacteria in 1.5 ml autoclaved eppendorfmicrofuge (1 ml/microfuge) was span down at 5000 rpm for 5 min, removedthe spent medium, pelleted bacteria was resuspended in 15% glycerolcontained P. acnes culture medium (1 ml/microfuge) without substrate andstored at −80° C. for future use.

For further P. acnes culture, one aliquoted P. acnes stock was taken outfrom −80° C. and thawed in ice. Almost 30 μl of P. acnes stock (1×10⁶/30μl) was added into 1 ml of culture medium with lipase substrate in each2 ml Eppendorf microfuge and incubated at 37° C. for 48 h in anaerobiccondition.

Efficacy Testing or Lipase Inhibitors Against P. acnes:

Orlistat or agent 767 (stocks 200 mM in ethanol) added at the indicatedconcentrations at the beginning of the incubations. At the end of theincubation period, a portion of the well mixed medium was removed, andfree fatty acid generation using the HR Series NEFA-HR(2) kit from WakoDiagnostics (Richmond Va.).

P. acnes Agar Culture

Spirit blue agar: Supplier, catalog #. Preparation, storage. Cultureinoculum size, culture conditions.

For plate preparation, 3.215 g of Spirit blue agar (Cat #M445, HiMediaLaboratories Pvt Ltd, Mumbai, India) dissolved in 100 ml of doubledistilled water, autoclaved, allowed to cool to 55° C., added 3 ml ofwarm lipase substrate and mixed well. In warm condition, the mixture waspoured into tissue culture plates (6 well, 12 well plates) inaseptically. The solidified agar plates were wrapped with parafilm andkept at 37° C. overnight to confirm no contamination. The extra plateswere kept at 4° C. freezer for future use. Either thawed P. acnes −80°C. stock was spread out (30 μl/well) in 6 well/12 well plates orinoculated throughout the well by means of inoculating loops. Then theplates were kept at 37° C. in BD GasPak EZ chamber with one anaerobesachets and waited for 5-7 days to see the bacterial colonies. Thesewere then visualized and photographed on a trans illuminator.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-19. (canceled)
 20. A method of treating organ failure in a subject inneed thereof, the method comprising administering to the subject atherapeutically effective amount of a compound selected from the groupconsisting of:

or a pharmaceutically acceptable salt thereof.
 21. The method of claim20, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 22. The method of claim20, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 23. The method of claim20, wherein the organ failure is renal failure.
 24. The method of claim20, wherein the organ failure is pulmonary failure.
 25. The method ofclaim 20, wherein the organ failure is multisystem organ failure. 26.The method of claim 25, wherein the organ failure is multiple organdysfunction syndrome involving at least the kidney and the lung.
 27. Themethod of claim 20, wherein the subject has acute pancreatitis.
 28. Themethod of claim 27, wherein the acute pancreatitis is severe.
 29. Themethod of claim 20, wherein the subject is a human subject.
 30. Themethod of claim 20, wherein the subject is obese.
 31. A method ofreducing the risk of organ failure in a subject suffering from acutepancreatitis, the method comprising administering to the subject atherapeutically effective amount of a compound selected from the groupconsisting of:

or a pharmaceutically acceptable salt thereof.
 32. The method of claim31, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 33. The method of claim31, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 34. The method of claim31, wherein the organ failure is renal failure.
 35. The method of claim31, wherein the organ failure is pulmonary failure.
 36. The method ofclaim 31, wherein the organ failure is multisystem organ failure. 37.The method of claim 36, wherein the organ failure is multiple organdysfunction syndrome involving at least the kidney and the lung.
 38. Themethod of claim 31, wherein the acute pancreatitis is severe.
 39. Themethod of claim 31, wherein the subject is a human subject.
 40. Themethod of claim 31, wherein the subject is obese.